Traditional approaches to tissue engineering have focused on biochemical cocktails to direct cells toward tissue-specific outcomes; in some cases mechanical forces have also been utilized. However, there is also a significant literature that details the role of biophysical signaling during tissue development and tissue regeneration, which has not yet been incorporated into the field of tissue engineering to date. The field of developmental biology has tracked the role of biophysical factors, such as membrane voltage potential and ion fluxes, during tissue regeneration, in wound healing, in embryonic patterning, and in many other critical tissue- related events. These data provide a clear link between membrane potential and cell behavior that determine tissue-specific outcomes. However, many molecular details are still unclear and this novel cell control modality has not been capitalized upon to advance tissue regeneration. The focus of the present proposal is to fill this void by specifically studying biophysical regulation of bone and adipose tissue regeneration, development and patterning. We will utilize 3D human tissue systems for bone and adipose tissue. The goal is to determine the utility of biophysical factors, such as membrane potential, on tissue-specific outcomes in the context of tissue regeneration in Aim #1, tissue development in Aim #2, and tissue patterning in Aim #3. We will compare the role of membrane potential during tissue regeneration and formation to the use of traditional biochemical cocktails as the controls. In the last aim, we will focus on spatial control of tissue outcomes via light-activated regulation of ion transport, mediated via a 3D optically-addressable scaffold system, to generate tissue patterns in vitro, analogous to morphological control during limb development. The outcome of the proposed study will be an entirely new approach to the regulation of tissue formation in vitro, with implications in many areas of regenerative medicine. Understanding and exploiting the role of bioelectrical signals on tissue outcomes in non-excitable cells will provide new insight into fundamental control of tissue regeneration, as well as novel approaches toward generating complex pattern development in tissues both in vitro and in vivo. PUBLIC HEALTH RELEVANCE: Exploitation of biophysical control of tissue regeneration is virtually unexplored territory in the field of tissue engineering, despite extensive studies in developmental biology that have clearly shown the importance of changes in membrane potential and endogenous electric fields during tissue/organ development and regeneration. Thus, the goal of this program is to determine the impact of membrane potential-regulated signaling on bone and adipose tissue regeneration, formation, and patterning. The outcome of this program would be an entirely new approach to tissue formation and control in vitro, with major implications for regeneration in vivo. Rational modulation of these powerful biophysical controls, with or without more traditional biochemical controls, will allow greater control of tissue development and function. Building upon principles from developmental biology, progress from the proposed studies will have a profound impact on the field of tissue engineering. Understanding the role of biophysical factors on tissue behavior will yield insight into fundamental control mechanisms underlying tissue growth and regeneration, offering a new perspective to the current tissue engineering paradigm. Such an understanding will also define a set of well-characterized pharmacological and molecular-genetic tools to enable novel approaches to complex tissue patterning.